scholarly journals An "individualist" model of an active genome in a developing embryo

2021 ◽  
Author(s):  
Shao-Kuei Huang ◽  
Sayantan Dutta ◽  
Peter H Whitney ◽  
Stanislav Shvartsman ◽  
Christine Rushlow
Keyword(s):  
Rna Polymerase Ii ◽  
Physical Distance ◽  
Drosophila Embryo ◽  
Drosophila Genome ◽  
Pol Ii ◽  
Individual Gene ◽  
Early Drosophila Embryo ◽  
Primary Driver ◽  
Genome Activation ◽  
Active Genes

The early Drosophila embryo provides unique experimental advantages for addressing fundamental questions of gene regulation at multiple levels of organization, from individual gene loci to the whole genome. Using Drosophila embryos undergoing the first wave of genome activation, we detected discrete "speckles" of RNA Polymerase II (Pol II), and showed that they overlap with transcribing loci. We characterized the spatial distribution of Pol II speckles and quantified how this distribution changes in the absence of the primary driver of Drosophila genome activation, the pioneer factor Zelda. Although the number and size of Pol II speckles were reduced, indicating that Zelda promotes Pol II speckle formation, we observed a uniform distribution of distances between active genes in the nuclei of both wildtype and zelda mutant embryos. This suggests that the topologically associated domains identified by Hi-C studies do little to spatially constrain groups of transcribed genes at this time. We provide evidence that linear genomic distance between transcribed genes is the primary determinant of measured physical distance between the active loci. Furthermore, we show active genes can have distinct Pol II pools even if the active loci are in close proximity. In contrast to the emerging model whereby active genes are clustered to facilitate co-regulation and sharing of transcriptional resources, our data support an "individualist" model of gene control at early genome activation in Drosophila. This model is in contrast to a "collectivist" model where active genes are spatially clustered and share transcriptional resources, motivating rigorous tests of both models in other experimental systems.

scholarly journals The miR-430 locus with extreme promoter density is a transcription body organizer, which facilitates long range regulation in zygotic genome activation

2021 ◽  
Author(s):  
Yavor Hadzhiev ◽  
Lucy Wheatley ◽  
Ledean Cooper ◽  
Federico Ansaloni ◽  
Celina Whalley ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Gene Cluster ◽  
Core Promoter ◽  
Single Copy ◽  
Early Embryo ◽  
Zygotic Genome Activation ◽  
Pol Ii ◽  
A Minor ◽  
Genome Activation ◽  
Zygotic Genome

In anamniote embryos the major wave of zygotic genome activation (ZGA) starts during the mid-blastula transition. This major wave of ZGA is facilitated by several mechanisms, including dilution of repressive maternal factors and accumulation of activating transcription factors during the fast cell division cycles preceding the mid-blastula transition. However, a set of genes escape global genome repression and are activated substantially earlier, during what is called, the minor wave of genome activation. While the mechanisms underlying the major wave of genome activation have been studied extensively, the minor wave of genome activation is little understood. In zebrafish the earliest expressed RNA polymerase II (Pol II) transcribed genes are activated in a pair of large transcription bodies depleted of chromatin, abundant in elongating Pol II and nascent RNAs (Hadzhiev et al., 2019; Hilbert et al., 2021). This transcription body includes the miR-430 gene cluster required for maternal mRNA clearance. Here we explored the genomic, chromatin organisation and cis-regulatory mechanisms of the minor wave of genome activation occurring in the transcription body. By long read genome sequencing we identified a remarkable cluster of miR-430 genes with over 300 promoters and spanning 0.6 Mb, which represent the highest promoter density of the genome. We demonstrate that the miR-430 gene cluster is required for the formation of the transcription body and acts as a transcription organiser for minor wave activation of a set of zinc finger genes scattered on the same chromosome arm, which share promoter features with the miR-430 cluster. These promoter features are shared among minor wave genes overall and include the TATA-box and sharp transcription start site profile. Single copy miR-430 promoter transgene reporter experiments indicate the importance of promoter-autonomous mechanisms regulating escape from global repression of the early embryo. These results together suggest that formation of the transcription body in the early embryo is the result of high promoter density coupled to a minor wave-specific core promoter code for transcribing key minor wave ZGA genes, which are required for the overhaul of the transcriptome during early embryonic development.

scholarly journals GAF is essential for zygotic genome activation and chromatin accessibility in the early Drosophila embryo

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Marissa M Gaskill ◽  
Tyler J Gibson ◽  
Elizabeth D Larson ◽  
Melissa M Harrison
Keyword(s):  
Chromatin Accessibility ◽  
Drosophila Embryo ◽  
Gaga Factor ◽  
Zygotic Transcription ◽  
Early Drosophila Embryo ◽  
Zygotic Gene ◽  
Pioneer Factor ◽  
Pioneer Factors ◽  
Genome Activation ◽  
Development Proceeds

Following fertilization, the genomes of the germ cells are reprogrammed to form the totipotent embryo. Pioneer transcription factors are essential for remodeling the chromatin and driving the initial wave of zygotic gene expression. In Drosophila melanogaster, the pioneer factor Zelda is essential for development through this dramatic period of reprogramming, known as the maternal-to-zygotic transition (MZT). However, it was unknown whether additional pioneer factors were required for this transition. We identified an additional maternally encoded factor required for development through the MZT, GAGA Factor (GAF). GAF is necessary to activate widespread zygotic transcription and to remodel the chromatin accessibility landscape. We demonstrated that Zelda preferentially controls expression of the earliest transcribed genes, while genes expressed during widespread activation are predominantly dependent on GAF. Thus, progression through the MZT requires coordination of multiple pioneer-like factors, and we propose that as development proceeds control is gradually transferred from Zelda to GAF.

scholarly journals What defines the maternal transcriptome?

2021 ◽  
Author(s):  
László Tora ◽  
Stéphane D. Vincent
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Protein Translation ◽  
Sole Source ◽  
Oocyte Growth ◽  
Pol Ii ◽  
Maternal Mrna ◽  
Protein Pool ◽  
Maternal Transcripts ◽  
Genome Activation

In somatic cells, RNA polymerase II (Pol II) transcription initiation starts by the binding of the general transcription factor TFIID, containing the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs), to core promoters. However, in growing oocytes active Pol II transcription is TFIID/TBP-independent, as during oocyte growth TBP is replaced by its vertebrate-specific paralog TBPL2. TBPL2 does not interact with TAFs, but stably associates with TFIIA. The maternal transcriptome is the population of mRNAs produced and stored in the cytoplasm of growing oocytes. After fertilization, maternal mRNAs are inherited by the zygote from the oocyte. As transcription becomes silent after oocyte growth, these mRNAs are the sole source for active protein translation. They will participate to complete the protein pool required for oocyte terminal differentiation, fertilization and initiation of early development, until reactivation of transcription in the embryo, called zygotic genome activation (ZGA). All these events are controlled by an important reshaping of the maternal transcriptome. This procedure combines cytoplasmic readenylation of stored transcripts, allowing their translation, and different waves of mRNA degradation by deadenylation coupled to decapping, to eliminate transcripts coding for proteins that are no longer required. The reshaping ends after ZGA with an almost total clearance of the maternal transcripts. In the past, the murine maternal transcriptome has received little attention but recent progresses have brought new insights into the regulation of maternal mRNA dynamics in the mouse. This review will address past and recent data on the mechanisms associated with maternal transcriptome dynamic in the mouse.

scholarly journals The pioneer factor GAF is essential for zygotic genome activation and chromatin accessibility in the early Drosophila embryo

2020 ◽  
Author(s):  
Marissa M. Gaskill ◽  
Tyler J. Gibson ◽  
Elizabeth D. Larson ◽  
Melissa M. Harrison
Keyword(s):  
Transcriptional Control ◽  
Chromatin Accessibility ◽  
Drosophila Embryo ◽  
Gaga Factor ◽  
Zygotic Transcription ◽  
Early Drosophila Embryo ◽  
Zygotic Gene ◽  
Pioneer Factor ◽  
Pioneer Factors ◽  
Genome Activation

AbstractFollowing fertilization, the genomes of the germ cells are reprogrammed to form the totipotent embryo. Pioneer transcription factors are required for remodeling the chromatin and driving the initial wave of zygotic gene expression. In Drosophila melanogaster, the pioneer factor Zelda is essential for development through this dramatic period of reprogramming, known as the maternal- to-zygotic transition (MZT). However, it was unknown whether additional pioneer factors were necessary for this transition. We identified an additional maternally encoded factor required for development through the MZT, GAGA Factor (GAF). GAF is needed to activate widespread zygotic transcription and to remodel the chromatin accessibility landscape. We demonstrated that Zelda preferentially controls expression of the earliest transcribed genes, while genes expressed during widespread activation are predominantly dependent on GAF. Thus, progression through the MZT requires coordination of multiple pioneer factors, and we propose that as development proceeds transcriptional control is gradually transferred from Zelda to GAF.

scholarly journals Transcription and splicing dynamics during early Drosophila development

RNA ◽  
2021 ◽  
pp. rna.078933.121
Author(s):  
Pedro Prudencio ◽  
Rosina Savisaar ◽  
Kenny Rebelo ◽  
Rui Goncalo Martinho ◽  
Maria Carmo-Fonseca
Keyword(s):  
Rna Polymerase Ii ◽  
Developmental Stages ◽  
Early Embryo ◽  
Drosophila Embryo ◽  
Dynamic Features ◽  
Nascent Transcript ◽  
Pol Ii ◽  
Cleavage And Polyadenylation ◽  
Dna Template ◽  
Early Developmental

Widespread co-transcriptional splicing has been demonstrated from yeast to human. However, most studies to date addressing the kinetics of splicing relative to transcription used either Saccharomyces cerevisiae or metazoan cultured cell lines. Here, we adapted native elongating transcript sequencing technology (NET-seq) to measure co-transcriptional splicing dynamics during the early developmental stages of Drosophila melanogaster embryos. Our results reveal the position of RNA polymerase II (Pol II) when both canonical and recursive splicing occur. We found heterogeneity in splicing dynamics, with some RNAs spliced immediately after intron transcription, whereas for other transcripts no splicing was observed over the first 100 nucleotides of the downstream exon. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. We studied the splicing dynamics of both nascent pre-mRNAs transcribed in the early embryo, which have few and short introns, as well as pre-mRNAs transcribed later in embryonic development, which contain multiple long introns. As expected, we found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. We further observed that genes transcribed in the early embryo tend to be isolated in the genome whereas genes transcribed later are often overlapped by a neighboring convergent gene. In isolated genes, transcription termination occurred soon after the polyadenylation site, while in overlapped genes Pol II persisted associated with the DNA template after cleavage and polyadenylation of the nascent transcript. Taken together, our data unravels novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo.

scholarly journals Continuous transcription initiation guarantees robust repair of transcribed genes and regulatory regions in response to DNA damage

2019 ◽  
Author(s):  
Anastasios Liakos ◽  
Dimitris Konstantopoulos ◽  
Matthieu D. Lavigne ◽  
Maria Fousteri
Keyword(s):  
Dna Damage ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
De Novo ◽  
Transcriptional Response ◽  
Genotoxic Stress ◽  
Chromatin Accessibility ◽  
Initiation Rate ◽  
Pol Ii ◽  
Active Genes

ABSTRACTInhibition of RNA synthesis caused by DNA damage-impaired RNA polymerase II (Pol II) elongation is found to conceal a local increase in de novo transcription, slowly progressing from Transcription Start Sites (TSSs) to gene ends. Although associated with accelerated repair of Pol II-encountered lesions and limited mutagenesis, it is still unclear how this mechanism is maintained during recovery from genotoxic stress. Here we uncover a surprising widespread gain in chromatin accessibility and preservation of the active histone mark H3K27ac after UV-irradiation. We show that the concomitant increase in Pol II release from promoter-proximal pause (PPP) sites of most active genes, PROMoter uPstream Transcripts (PROMPTs) and enhancer RNAs (eRNAs) favors unrestrained initiation, as demonstrated by the synthesis of short nascent RNAs, including TSS-associated RNAs (start-RNAs). In accordance, drug-inhibition of the transition into elongation replenished the post-UV reduced levels of pre-initiating pol II at TSSs. Continuous engagement of new Pol II thus ensures maximal transcription-driven DNA repair of active genes and non-coding regulatory loci. Together, our results reveal an unanticipated layer regulating the UV-triggered transcriptional-response and provide physiologically relevant traction to the emerging concept that transcription initiation rate is determined by pol II pause-release dynamics.

scholarly journals Association of SARFH (sarcoma-associated RNA-binding fly homolog) with regions of chromatin transcribed by RNA polymerase II.

1995 ◽  
Vol 15 (8) ◽  
pp. 4562-4571 ◽  
Author(s):  
D Immanuel ◽  
H Zinszner ◽  
D Ron
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Polytene Chromosomes ◽  
Drosophila Embryo ◽  
Cdna Clones ◽  
Rna Pol Ii ◽  
Pol Ii

Many oncogenes associated with human sarcomas are composed of a fusion between transcription factors and the N-terminal portions of two similar RNA-binding proteins, TLS and EWS. Though the oncogenic fusion proteins lack the RNA-binding domain and do not bind RNA, the contribution from the N-terminal portion of the RNA-binding protein is essential for their transforming activity. TLS and EWS associate in vivo with RNA polymerase II (Pol II) transcripts. To learn more about the target gene specificity of this interaction, the localization of a Drosophila melanogaster protein that has extensive sequence identity to the C-terminal RNA-binding portions of TLS and EWS was studied in preparations of Drosophila polytene nuclei. cDNA clones encoding the full-length Drosophila TLS-EWS homolog, SARFH (stands for sarcoma-associated RNA-binding fly homolog), were isolated. Functional similarity to TLS and EWS was revealed by the association of SARFH with Pol II transcripts in mammalian cells and by the ability of SARFH to elicit homologous down-regulation of the levels of the mammalian proteins. The SARFH gene is expressed in the developing Drosophila embryo from the earliest stages of cellularization and is subsequently found in many cell types. In preparations of polytene chromosomes from salivary gland nuclei, SARFH antibodies recognize their target associated with the majority of active transcription units, revealed by colocalization with the phosphorylated form of RNA Pol II. We conclude that SARFH and, by homology, EWS and TLS participate in a function common to the expression of most genes transcribed by RNA Pol II.

scholarly journals Cotranscriptional Recruitment of the U1 snRNP to Intron-Containing Genes in Yeast

2003 ◽  
Vol 23 (16) ◽  
pp. 5768-5779 ◽  
Author(s):  
Kimberly M. Kotovic ◽  
Daniel Lockshon ◽  
Lamia Boric ◽  
Karla M. Neugebauer
Keyword(s):  
Rna Polymerase Ii ◽  
Splicing Factors ◽  
Promoter Regions ◽  
Pol Ii ◽  
Intronless Genes ◽  
U1 Snrnp ◽  
Intron Removal ◽  
Capping Enzymes ◽  
Active Genes

ABSTRACT Evidence that pre-mRNA processing events are temporally and, in some cases, mechanistically coupled to transcription has led to the proposal that RNA polymerase II (Pol II) recruits pre-mRNA splicing factors to active genes. Here we address two key questions raised by this proposal: (i) whether the U1 snRNP, which binds to the 5′ splice site of each intron, is recruited cotranscriptionally in vivo and, (ii) if so, where along the length of active genes the U1 snRNP is concentrated. Using chromatin immunoprecipitation (ChIP) in yeast, we show that elevated levels of the U1 snRNP were specifically detected in gene regions containing introns and downstream of introns but not along the length of intronless genes. In contrast to capping enzymes, which bind directly to Pol II, the U1 snRNP was poorly detected in promoter regions, except in genes harboring promoter-proximal introns. Detection of the U1 snRNP was dependent on RNA synthesis and was abolished by intron removal. Microarray analysis revealed that intron-containing genes were preferentially selected by ChIP with the U1 snRNP. Thus, U1 snRNP accumulation at genes correlated with the presence and position of introns, indicating that introns are necessary for cotranscriptional U1 snRNP recruitment and/or retention.

scholarly journals A hyperactive transcriptional state marks genome reactivation at the mitosis-G1 transition

2016 ◽  
Author(s):  
Chris C.-S. Hsiung ◽  
Caroline Bartman ◽  
Peng Huang ◽  
Paul Ginart ◽  
Aaron J. Stonestrom ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Mammalian Cells ◽  
Transcriptional Activity ◽  
De Novo ◽  
Pol Ii ◽  
Mature Mrna ◽  
Genome Wide ◽  
Transcriptional State ◽  
Active Genes

AbstractDuring mitosis, RNA polymerase II (Pol II) and many transcription factors dissociate from chromatin, and transcription ceases globally. Transcription is known to restart in bulk by telophase, but whether de novo transcription at the mitosis-G1 transition is in any way distinct from later in interphase remains unknown. We tracked Pol II occupancy genome-wide in mammalian cells progressing from mitosis through late G1. Unexpectedly, during the earliest rounds of transcription at the mitosis-G1 transition, ~50% of active genes and distal enhancers exhibit a spike in transcription, exceeding levels observed later in G1 phase. Enhancer-promoter chromatin contacts are depleted during mitosis and restored rapidly upon G1 entry, but do not spike. Of the chromatin-associated features examined, histone H3 lysine 27 acetylation levels at individual loci in mitosis best predict the mitosis-G1 transcriptional spike. Single-molecule RNA imaging supports that the mitosis-G1 transcriptional spike can constitute the maximum transcriptional activity per DNA copy throughout the cell division cycle. The transcriptional spike occurs heterogeneously and propagates to cell-to-cell differences in mature mRNA expression. Our results raise the possibility that passage through the mitosis-G1 transition might predispose cells to diverge in gene expression states.

scholarly journals RNA polymerase II interacts with the Hspa1b promoter in mouse epididymal spermatozoa

Reproduction ◽  
2009 ◽  
Vol 137 (6) ◽  
pp. 923-929 ◽  
Author(s):  
Donald C Wilkerson ◽  
Kevin D Sarge
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Stage ◽  
Western Blots ◽  
Pol Ii ◽  
Epididymal Spermatozoa ◽  
The One ◽  
Genome Activation ◽  
Embryonic Viability ◽  
Zygotic Genome

TheHspa1b(Hsp70.1) gene is one of the first genes expressed after fertilization, with expression occurring during the minor zygotic genome activation (ZGA) in the absence of stress. This expression can take place in the male pronucleus as early as the one-cell stage of embryogenesis. The importance of HSPA1B for embryonic viability during times of stress is supported by studies showing that depletion of this protein results in a significant reduction in embryos developing to the blastocyte stage. Recently, we have begun addressing the mechanism responsible for allowing expression ofHspa1bduring the minor ZGA and found that heat shock transcription factor (HSF) 1 and 2 bind theHspa1bpromoter during late spermatogenesis. In this report, we have extended those studies using western blots and chromatin immunoprecipitation assays and found that RNA polymerase II (Pol II) is present in epididymal spermatozoa and bound to theHspa1bpromoter. These present results, in addition to our previous results, support a model in which the binding of HSF1, HSF2, SP1, and Pol II to the promoter ofHspa1bwould allow the rapid formation of a transcription-competent state during the minor ZGA, thereby allowingHspa1bexpression.

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